High-density lipoprotein (HDL) is one of lipoproteins, and it has a specific gravity from 1.063 to 1.210. Cholesterol in HDL (HDL-C) has been known as a negative risk factor for coronary heart disease (CHD). In recent years, there has been an increasing interest in the clinical significance of HDL subfractions, HDL2 and HDL3. HDL2 is a lipoprotein having a specific gravity from 1.063 to 1.125, whereas HDL3 is a lipoprotein having a specific gravity from 1.125 to 1.210.
Nascent HDL secreted from the liver or the intestine adheres to the peripheral cell membrane, and it absorbs free cholesterol therefrom. The thus absorbed free cholesterol is converted to esterified cholesterol by the action of LCAT (lecithin cholesterol acyltransferase) that is present on the surface of HDL, and then, spherical HDL3 is formed having this esterified cholesterol as a core. Then, the amount of the esterified cholesterol is increased in HDL3 by the action of LCAT, and as a result, HDL3 is converted to HDL2.
Cholesterol in HDL2 is metabolized by two pathways. One is a pathway by which cholesterol in HDL2 is directly incorporated into the liver in a state of HDL2 as such and is then excreted as a bile acid. The other is a pathway, by which esterified cholesterol in HDL2 is exchanged with triglycerides contained in triglycerides-rich lipoproteins, such as a very low-density lipoprotein (VLDL), an intermediate density lipoprotein (IDL) and a low-density lipoprotein (LDL), by the action of CETP (cholesterol ester transfer protein), and the esterified cholesterol are transported to the triglycerides-rich lipoproteins (reverse cholesterol transport system).
As a method for measuring cholesterol in an HDL subfraction, a precipitation method comprising a step of separating HDL3 from HDL2 and a homogeneous method that does not comprise a step of separating HDL3 from HDL2 have been known so far.
As a precipitation method, a method for measuring cholesterol in HDL3 contained in a sample by a single separation operation using heparin, divalent metal ions and dextran sulfate (for example, Patent Document 1 and Non-patent Document 1), a method for measuring cholesterol in HDL3 contained in a sample by a double separation operation (for example, Non-patent Documents 2 to 4), and the like have been known. The method for measuring cholesterol in HDL3 contained in a sample by the single separation operation is a method comprising agglutinating lipoproteins in a sample, other than HDL3, then separating and removing them, and then measuring cholesterol in the thus obtained HDL3. The method for measuring cholesterol in HDL3 contained in a sample by the double separation operation is a method comprising first agglutinating lipoproteins in a sample, other than HDL, then removing lipoproteins other than HDL by centrifugation, then agglutinating HDL2 in the obtained supernatant containing HDL, then removing the HDL2 by centrifugation, then recovering HDL3 contained in the supernatant, and then measuring cholesterol in the thus obtained HDL3.
As a homogeneous method, a method using an enzyme exhibiting high specificity to HDL and a nonionic surfactant having an HLB value of 17 or greater has been known (for example, Patent Document 2).
There is a need for a method for simply and precisely measuring cholesterol in an HDL subfraction contained in a sample without performing complicated operations such as centrifugation.